Study of the biochemical changes in the cytoplasm of in vitro matured bovine oocytes in media enriched with Vascular Endothelial Growth Factor-D by Raman microscopy

Abstract

In recent years, numerous studies have focused on the optimization of in vitro embryoproduction (IVP) of animals of productive interest, including bovines. In vitro maturation(IVM) of oocytes is the first stage of the IVP and can determine the performance of theprocess and the quality of the future embryo. Therefore, the optimization of the IVM mediawould make it possible to increase the number of competent oocytes. The aim of this workis to evaluate the impact of vascular endothelial growth factor-D (VEGF-D) on thecytoplasm of bovine oocytes during the IVM, by Raman microscopy.The spectra were taken from the cytoplasm of oocytes that were subjected to the IVMprocess for 18 and 22 h in the absence (control: 18H and 22H) and in the presence of thefactor (18H-F and 22H-F). For a more precise analysis of cytoplasmic changes during IVM,the zona pellucida was removed from the oocytes by enzymatic digestion. The averagespectrum of the 18H-F group showed a marked increase in the intensities of thecharacteristic protein bands with respect to the 18H spectrum. When the spectra thatcompleted the maturation time (22h) were related to each other, only subtle changes inintensity were observed in some bands assigned to amino acids. A principal componentanalysis (PCA), multivariate statistical calculation, was implemented for the signals thatshowed the greatest spectral difference. The scatter graph obtained by the PCA revealedthat the oocytes of groups 18H-F and 22H-F are grouped in the sector of the graphrepresenting the presence of intense characteristic protein bands. We previouslydemonstrated that, throughout IVM, there is a gradual accumulation of proteins in thecytoplasm of bovine oocytes, being greater at the end of the process (22h). All these dataconfirm that the supplementation of the maturation media with VEGF-D increases theprotein content in the cytoplasm of bovine oocytes and, therefore, could have afavourable impact on the performance of IVP.